View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central International Journal of Systematic and Evolutionary Microbiology (2010), 60, 1527–1531 DOI 10.1099/ijs.0.015016-0 Enterorhabdus caecimuris sp. nov., a member of the family Coriobacteriaceae isolated from a mouse model of spontaneous colitis, and emended description of the genus Enterorhabdus Clavel et al. 2009 Thomas Clavel,1 Wayne Duck,2 Ce´dric Charrier,3 Mareike Wenning,4 Charles Elson2 and Dirk Haller1 Correspondence 1Biofunctionality, ZIEL – Research Center for Nutrition and Food Science, Technische Universita¨t Thomas Clavel Mu¨nchen (TUM), 85350 Freising Weihenstephan, Germany [email protected] 2Division of Gastroenterology and Hepatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 3NovaBiotics Ltd, Aberdeen, AB21 9TR, UK 4Microbiology, ZIEL – Research Center for Nutrition and Food Science, TUM, 85350 Freising Weihenstephan, Germany The C3H/HeJBir mouse model of intestinal inflammation was used for isolation of a Gram- positive, rod-shaped, non-spore-forming bacterium (B7T) from caecal suspensions. On the basis of partial 16S rRNA gene sequence analysis, strain B7T was a member of the class Actinobacteria, family Coriobacteriaceae, and was related closely to Enterorhabdus mucosicola T T Mt1B8 (97.6 %). The major fatty acid of strain B7 was C16 : 0 (19.1 %) and the respiratory quinones were mono- and dimethylated. Cells were aerotolerant, but grew only under anoxic conditions. Strain B7T did not convert the isoflavone daidzein and was resistant to cefotaxime. The results of DNA–DNA hybridization experiments and additional physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain B7T from the type strain of E. mucosicola. Therefore, strain B7T represents a novel species, for which the name Enterorhabdus caecimuris sp. nov. is proposed. The type strain is B7T (5DSM 21839T 5CCUG 56815T). The family Coriobacteriaceae currently comprises 13 caecum of a C3H/HeJBir mouse, a mouse substrain prone genera, four of which have been described recently, and to spontaneous colitis (Sundberg et al., 1994), after 3 days includes Adlercreutzia equolifaciens (Maruo et al., 2008), growth at 37 uC on ATCC medium 602 E. Additional the type species of which was isolated from human faeces; information on strain isolation and 16S rRNA gene Asaccharobacter celatus (Minamida et al., 2008), isolated sequencing has been published elsewhere (Duck et al., from a rat caecum; Enterorhabdus mucosicola (Clavel et al., 2007). Unless otherwise stated, all experiments for the 2009), isolated from the inflamed ileal mucosa of a mouse; description of strain B7T were carried out as described and Gordonibacter pamelaeae (Wu¨rdemann et al., 2009), previously (Clavel et al., 2009). Bacteroides vulgatus was isolated from a patient with Crohn’s disease. used as a positive control for the determination of growth with bile salts (no. 48305; Fluka). Cellular fatty acids, During the course of experiments focused on flagellated respiratory quinones, peptidoglycan and whole-cell sugars bacteria and their implication in intestinal inflammation T were analysed by the DSMZ, Braunschweig, Germany, (Duck et al., 2007), strain B7 was isolated from the according to standard procedures (Sasser, 1990; Cashion et al., 1977; De Ley et al., 1970; Huß et al., 1983; Mesbah Abbreviation: FT-IRS, Fourier-transform infrared spectroscopy. et al., 1989; Rhuland et al., 1955; Staneck & Roberts, 1974; The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and Tamaoka & Komagata, 1984; Visuvanathan et al., 1989; T gyrase B gene sequences of strain B7 are DQ789120 and Whiton et al., 1985). GQ409830, respectively. T Supplementary figures showing the polar lipid compositions and daidzein 16S rRNA gene sequences from strain B7 (determined as conversion abilities of strain B7T and the type strain of Enterorhabdus described previously; Duck et al., 2007) and GenBank were mucosicola are available with the online version of this paper. aligned using BioEdit version 7.0.5.3 (Hall, 1999) and a 015016 G 2010 IUMS Printed in Great Britain 1527 T. Clavel and others rooted tree was constructed using the neighbour-joining 16S rRNA gene sequence similarity values with members of method with CLUSTAL X version 1.8. Bootstrap values were these genera of ,94 %. calculated on the basis of 1000 resamplings. The max- The gyrase B gene of strain B7T was amplified as described imum-parsimony method was used to confirm the previously (Santos & Ochman, 2004). Amplicons topology of the phylogeny. Fig. 1 gives a current (1500 bp) were purified using agarose gel electrophoresis phylogenetic overview of the family Coriobacteriaceae and and the Wizard SV Gel and PCR Clean-Up System the position of strain B7T. Sequence similarity values were (Promega). Purified products were sequenced using the obtained with the DNA distance matrix function in the primers gyrBBNDN1 (59-CCGTCCACGTCGGCRTCNG- BioEdit software. The 16S rRNA gene sequence of strain YCAT-39) and gyrBBAUP2 (59-GCGGAAGCGGCCNGSN- B7T (1336 bp) was related most closely (.99 %) to ATGTA-39). The gyrase B gene sequence of strain B7T sequences originating from as-yet-uncultured mouse shared 95.7 and 79.3 % similarity with sequences from E. intestinal bacteria (Ley et al., 2005, 2006) and E. mucosicola mucosicola Mt1B8T (GenBank accession no. EU594341) and Mt1B8T (97.6 %). Lower similarities were found to Eggerthella lenta DSM 2243T (EU594342; 524 bp), respec- sequences from Asaccharobacter celatus do03T (93.4 %), tively. Strain B7T exhibited low DNA–DNA relatedness to E. Adlercreutzia equolifaciens FJC-B9T (93.3 %) and two mucosicola DSM 19490T (28.0±2.0 %, two experiments), Eggerthella strains (,90 %). The resolution of 16S rRNA which supported the fact that these two bacteria belong to gene sequence analysis does not allow the identification of different species. The DNA G+C content of strain B7T closely related species. However, it consistently depicts (64.5 mol%) was comparable to those reported in the phylogenetic relationships from the level of domains to literature for its phylogenetic neighbours. moderately related species (Stackebrandt & Goebel, 1994). It has been proposed that a genus could be defined as The results of phenotypic and chemotaxonomic analyses containing species that have 95 % 16S rRNA gene sequence are given in the species description and in Table 1. The similarity to each other (Rossello-Mora & Amann, 2001). fatty acid profile of strain B7T was similar to that of E. We suggest that strain B7T does not belong to either of the mucosicola DSM 19490T. The diamino acid in the genera Adlercreutzia or Asaccharobacter, as the strain has peptidoglycan was identified as meso-diaminopimelic acid, Fig. 1. Phylogenetic position of strain B7T within the family Coriobacteriaceae, based on a neighbour-joining analysis of 16S rRNA gene sequences (1338 bp). Bootstrap values based on 1000 resamplings are shown at branch nodes. Hash signs indicate that the corres- ponding nodes were also recovered in the tree generated using the maximum-parsimony method. Eubacterium limosum ATCC 8486T, a member of the phylum Firmicutes, was used as an outgroup. Bar, 10 substitutions per 100 nucleotide positions. 1528 International Journal of Systematic and Evolutionary Microbiology 60 Enterorhabdus caecimuris sp. nov. Table 1. Characteristics that differentiate strain B7T and Table 1. cont. E. mucosicola DSM 19490T Characteristic 1 2 Strains: 1, B7T;2,E. mucosicola DSM 19490T. Data were taken from this study. DMA, Dimethylacetal; DMMK, dimethylmenaquinone; C18 : 1 c9 15.25 16.68 21 MMK, monomethylmenaquinone; R, resistant (MIC .32 mgml ); C18 : 1 c9 DMA 4.94 4.75 ND, not determined. C18 : 1 c11 DMA 0.59 0.56 C18 : 1 c11/t9/t6 4.16 3.83 C c9,12 1.24 1.30 Characteristic 1 2 18 : 2 Diaminopimelic acid meso LL *Data obtained with Rapid ID32A identification system for anaerobes Major menaquinones (%) (bioMe´rieux). MMK-6 60 100 DValues are expressed as mean±SD of six replicates (three DMMK-6 40 0 independent experiments with duplicates). Whole-cell sugars Glucose + 2 Enzyme activities* which so far has been reported only for the peptidoglycan Aminopeptidase 2 + type A1c and three variations of peptidoglycan type A4c. Glutamic acid + 2 The quinones were monomethylmenaquinone-6 (60 %) decarboxylase and dimethylmenaquinone-6 (40 %). Isoflavone conversion T + For polar lipid analysis, batch cultures (1.5 l) of strain B7 Daidzein 2 T Genistein ND + and E. mucosicola DSM 19490 were grown under anoxic [ Antibiotic MIC (mgml21)D conditions for 48 h in GYBHIc brain–heart infusion broth 21 Cefotaxime R (.32) 1.250±0.112 (no. 211059; BD) supplemented with (l ) 4 g glucose, 4 g ] Ciprofloxacin 0.305±0.035 R (.32) yeast extract and 0.05 % (w/v) cysteine and harvested by Clarithromycin ,0.016 ,0.016 centrifugation [5525 g for 10 min at room temperature in Clindamycin 0.105±0.010 ,0.016 500 ml containers using a 4K15C centrifuge (Sigma)]. Erythromycin ,0.016 0.048±0.007 Pellets were resuspended in filter-sterilized PBS [(l distilled 21 Metronidazole 0.016±0.000 0.034±0.004 water) : 8.60 g NaCl, 0.87 g Na2HPO4, 0.40 g KH2PO4; Oxacillin R (36.000±5.750) 4.667±0.667 pH 7.2] and supernatants were centrifuged again as above. Tetracycline 0.120±0.005 0.115±0.007 Resuspended pellets were pooled in 50 ml Falcon tubes and Tobramycin 4.333±0.558 2.667±0.211 centrifuged as above for 15 min. Supernatants were Vancomycin 1.500±0.129 1.333±0.105 discarded first by inverting the tubes and subsequently Cellular fatty acids pipetting the remaining liquid after the tubes had been left iso-C12 : 0 0.36 0.50 to stand for 30 s. Samples were stored at 280 uC prior to C12 : 0 1.21 0.72 shipping on dry ice. Polar lipid analysis was done by the iso-C13 : 0 2 0.15 Identification Service of the DSMZ and Dr B.